HEMOLYTIC SYSTEMS CONTAINING ANIONIC DETERGENTS

1. The members of the homologous series of anionic detergents, the sodium salts of the sulfated straight chain alcohols with the general formula C_nH_2n+1·SO₃·Na, are hemolytic, the lytic activity being at a maximum when the compound contains 14 carbon atoms in the chain. In systems in which lysis is comparatively rapid, the hemolytic effect increases with increasing pH, but in systems containing quantities of lysin near the asymptotic concentrations the pH dependence of the activity is reversed. The effect of temperature is principally one on the velocity constant of the lytic reaction, with smaller effects on the position of the asymptotes of the time-dilution curves and on their shape. 2. The quantities of the detergents which produce disk-sphere transformations are approximately one-tenth of those required to produce complete hemolysis. In most cases, the shape change occurs when there are too few detergent molecules present to cover the red cell surfaces with a monolayer. 3. Plasma inhibits the hemolytic action of these detergents, and, in the quantities in which they occur in plasma, lecithin, serum globulin, cholesterol, and serum albumin, produce inhibitory effects which increase in that order in systems containing the C-14 sulfate. It can be inferred from these inhibitory effects that the anionic detergents can form compounds or complexes with lipid, lipoprotein, and protein components of the red cell ultrastructure.     

Hemolytic Activity of a Togavirus,Getah*

A purified toga-alphavirus, Getah (GET), showed optimal hemolytic activity for one-day-old chick red blood cells when incubated at 37 C for 120 min at pH 6.2. Experimental data obtained from various angles, such as pH dependency, inhibition by virus-specific antiserum and by concanavalin A, indicated that the hemolysis was a property of the virus particle itself. Although the mechanism of hemolysis by togaviruses has not been known, our results indicated that viral lipids may participate in this activity since the hemolytic activity was impaired by delipidation procedures.     

Delayed hemolysis of human erythrocytes in solutions of glucose

There has been described a type of hemolysis which occurs under certain defined conditions when erythrocytes are suspended in glucose solution. It consists of a prolytic phase lasting about an hour, followed by a hemolytic phase lasting about 2 hours. The physical factors controlling this delayed hemolysis have been investigated. The system is especially sensitive to changes of pH and of temperature. This type of hemolysis is inhibited by increased osmotic pressure and by phlorhizin, but not, as far as can be ascertained, by fluoride or iodoacetate. It is possible, but not yet proved, that delayed hemolysis in glucose solution is dependent on enzymic activity. Phosphorylation may be the limiting factor. During the prolytic phase the cells are easily permeable to potassium. It is concluded that the development of cation permeability is not a direct cause of hemolysis.     

The mechanism of the hemolytic activity of polyene antibiotics

The kinetics of the filipin-, amphotericin B- and nystatin-induced hemolysis of human erythrocytes were investigated. Filipin-induced hemolysis is of the damage type. It is an all-or-none process, partly inhibited by Ca2+ or Ba2+ but not by Mg2+, Na+ or SO42-. The hemolytic activity of filipin is explained by the formation of large aggregates within the erythrocyte membrane in the form of large perforations, permeable to substances of low molecular weight as well as to macromolecules, including hemoglobin. In isotonic KCl solution, both amphotericin B and nystatin, at low concentrations, form smaller aggregates within the membranes. As a result, the permeability of the membranes to KCl increases and hemolysis occurs. However, the kinetics of the hemolysis induced by the two polyenes is complex. The process shows some features of the permeability type and some of the damage type. It is suggested that amphotericin B and nystatin may simultaneously form a number of transport systems, differing in their molecular organisation and hemolytic activity. Their participation in erythrocyte membrane permeability can be modified by small changes in membrane organisation and the chemical composition of the incubation medium. In isotonic solutions of divalent cation chlorides, and at higher antibiotic concentration, additional aggregates, allowing divalent cations to permeate, appear. These structures do not permit SO4(2-) to permeate.     

Specific DNA recognition by the type II restriction endonuclease MunI: the effect of pH.

To investigate the effect of pH on sequence-specific binding, a thermodynamic characterization of the interaction of the protein MunI with a specific, and a nonspecific, oligonucleotide was performed. MunI is a type II restriction endonuclease which is able to bind specifically, but loses its enzymatic activity in the absence of magnesium ions. Comparison of the specific and nonspecific interactions at 10 and 25 degrees C shows that the latter is accompanied by a small change in enthalpy, and a negligible change in constant pressure heat capacity. On going through the pH range 5.75-9.0 at 25 degrees C, the affinity of specific complex formation is reduced by 20-fold. The interaction is accompanied by the protonation of groups assumed to be on the protein. Based on the simplest model that will fit the data, two distinct protonation events are observed. At low pH, two groups per protein molecule undergo protonation with a pK(a) of 6.0 and 6.9 in the free and bound forms, respectively. At high pH, a further independent protonation occurs involving two groups with pK(a) values of 8.9 and approximately 10.7 in the free and bound forms, respectively. The change in heat capacity ranges from -2.7 to -1.7 kJ mol(-1) K(-1) in going from pH 6.5 to 8.5. This range of variation of change in heat capacity can be accounted for by the effects of protonation of the interacting molecules. The change in heat capacity, calculated from surface area burial using a previously established relationship (1.15 kJ mol(-1) K(-1)), does not correlate well with the experimentally determined values.     

pH dependence of the circular dichroic bands of phenylmethanesulfonyl-mesentericopeptidase.

The effect of pH on the circular dichroism spectra of phenylmethanesulfonyl-mesentericopeptidase (peptidyl peptide hydrolase, EC 3.4.21) was studied. The ellipticity of the bands below 250 nm, which reflects the backbone conformation of the protein molecule, remains almost unchanged in the pH range 6.2--10.4. However, below pH 6.2 and above pH 10.4 a conformational transition occurs. The pH-dependent changes above 250 nm were also studied. The titration of the CD band at 296 nm reflects the ionization of the "exposed" tyrosines, which phenolic groups are fully accessible to the solvent. An apparent pK of 9.9 is calculated from the titration curve. It is concluded that ionization of the tyrosyl residues with normal pK's is complete before conformational changes in the protein molecule occur.     

Some properties of the alpha-hemolysin produced by a hemolytic strain of E. coli]

It was found that alpha-hemolysin of E. coli P 678 HIy+ was maximally active against human erythrocytes at pH 6.5. The hemolytic activity is characterized in time by a distinct lag-phase and a phase of the greatest velocity of the reaction immediately following it. The duration of the lag-phase and also the rate of hemolysis depends on alpha-hemolysin concentration, whose increase is accompanied by a decrease of the lag-phase and acceleration of hemolysis. There is a definite limit below which the duration of the lag-phase remains unchanged with further increase of hemolysin concentration. There was noted a linear relationship between the amount of erythrocytes taken for the test and the rate of hemoglobin release and also a temperature activation of the hemolytic reaction.     

Studies on the role of goat heart galectin-1 as an erythrocyte membrane perturbing agent

Galectins are β-galactoside binding lectins with a potential hemolytic role on erythrocyte membrane integrity and permeability. In the present study, goat heart galectin-1 (GHG-1) was purified and investigated for its hemolytic actions on erythrocyte membrane. When exposed to various saccharides, lactose and sucrose provided maximum protection against hemolysis, while glucose and galactose provided lesser protection against hemolysis. GHG-1 agglutinated erythrocytes were found to be significantly hemolyzed in comparison with unagglutinated erythrocytes. A concentration dependent rise in the hemolysis of trypsinized rabbit erythrocytes was observed in the presence of GHG-1. Similarly, a temperature dependent gradual increase in percent hemolysis was observed in GHG-1 agglutinated erythrocytes as compared to negligible hemolysis in unagglutinated cells. The hemolysis of GHG-1 treated erythrocytes showed a sharp rise with the increasing pH up to 7.5 which became constant till pH 9.5. The extent of erythrocyte hemolysis increased with the increase in the incubation period, with maximum hemolysis after 5 h of incubation. The results of this study establish the ability of galectins as a potential hemolytic agent of erythrocyte membrane, which in turn opens an interesting avenue in the field of proteomics and glycobiology.     

Copper sorption by chitosan in the presence of citrate ions: influence of metal speciation on sorption mechanism and uptake capacities

The presence of organic ligands in a solution containing metal ions modifies metal speciation, which in turn changes the sorption mechanism, optimum pH range and maximum sorption capacity. The present work investigates the sorption of copper by chitosan in the presence of citrate at different metal/ligand ratios. Copper uptake in acidic solution takes place through electrostatic attraction between the protonated amine groups of chitosan and anionic copper-citrate complexes (mainly Cu(OH)L2- but also a small fraction of CuL-). Sorption was negligible below pH 3 due to competition from dissociated anionic ligand and counter ions brought about by dissociation of the acid used for pH control. Actually, copper sorption begins to be significant when the fraction of anionic copper-complexes exceeds that of anionic copper-free ligand. So sorption capacity strongly increases up to pH 4.5-5.5. Above pH 5.5, the progressive decrease of amine protonation leads to a linear decrease in sorption capacity. An excess of ligand leads to an increase in the fraction of free dissociated (anionic) ligand that may compete for electrostatic attraction on protonated amine groups and therefore leads to a decrease in sorption capacities.     

Properties of phospholipase A2 from the venom of the large hornet Vespa orientalis

Some properties (catalytic and hemolytic activity, pH and temperature optima, stability, substrate specificity, effects of detergents and metal ions, N-terminal sequence, chemical modification of histidine in the enzyme active center, etc.) of phospholipase A2 from hornet (Vespa orientalis) venom were studied. It was shown that phospholipase A2 from hornet venom differs essentially from other enzymes of this species in terms of stability, catalytic properties and structural features. The active center of the enzyme contains an essential histidine residue, similar to other phospholipases A2 from various sources. Unlike other known forms of phospholipase A2, the enzyme under study exerts a pronounced hemolytic action. The hemolysis is inhibited by Ca2+ at concentrations capable of inducing the activation of the hydrolytic activity of the enzyme.     

Acid-base equilibria in rhodopsin: dependence of the protonation state of glu134 on its environment

Glutamic acid E134 in rhodopsin is part of a highly conserved triad, D(E)RY, located near the cytoplasmic lipid/water interface in transmembrane helix 3 of G protein-coupled receptors (GPCRs). A large body of experimental evidence suggests that the protonation of E134 plays a role in the mechanism of activation of rhodopsin and other GPCRs as well. For E134 to change its protonation state, its pK(a) value must shift from values below physiological pH to higher values. Because of the proximity of the triad to the lipid/water interface, it was hypothesized that a change in solvent around E134 from water to lipid could induce such a shift in pK(a). To test this hypothesis, the pK(a) values of the titratable amino acid residues in rhodopsin have been calculated and the change in solvent around E134 was modeled by shifting the position of the lipid/water interface. The approach used to carry out the pK(a) calculations takes into account the partial immersion of transmembrane proteins in lipid. Qualitative experimental evidence is available for several residues regarding their likely protonation state in rhodopsin at or near physiological pH. Comparison of the calculated pK(a) values with these experimental findings shows good agreement between the two. Notably, glutamic acids E122 and E181 were found to be protonated. The pK(a) values were then calculated for a range of lipid/water interface positions. Although the surrounding solvent of several titratable residues changed from water to lipid in this range, leading to pK(a) shifts in most cases, only for E134 would the shift lead to a change in protonation state at physiological pH. Thus, our results show that the protonation state of E134 is particularly sensitive to its environment. This sensitivity together with the location of E134 near the actual position of the lipid/water interface could be a strategic element in the mechanism of activation of rhodopsin.     

Stoichiometry of hemolysis by the polyene antibiotic lucensomycin

The stoichiometry of hemolysis by the polyene antibiotic lucensomycin was investigated. It appears that hemolysis occurs only when a relatively high fraction (probably between 15 and 40%) of the cholesterol sites in the erythrocyte membrane have combined with the polyene. Also in phospholipid-cholesterol vesicles the increase of permeability requires occupancy of 40–50% of the existing cholesterol sites.As for the possible cooperative effect in the hemolytic process, it is probable that several (at least 9–10) lucensomycin-cholesterol adducts must interact on each side of the membrane to form an aqueous channel; the distribution of these adducts in the erythrocyte membrane occurs, however, apparently at random.     

Chemical mechanism of the adenosine cyclic 3',5'-monophosphate dependent protein kinase from pH studies

The pH dependence of kinetic parameters and inhibitor dissociation constants for the adenosine cyclic 3',5'-monophosphate dependent protein kinase reaction has been determined. Data are consistent with a mechanism in which reactants selectively bind to enzyme with the catalytic base unprotonated and an enzyme group required protonated for peptide (Leu-Arg-Arg-Ala-Ser-Leu-Gly) binding. Binding of the peptide apparently locks both of the above enzyme residues in their correct protonation state. MgATP preferentially binds fully ionized and requires an enzyme residue (probably lysine) to be protonated. The maximum velocity and V/KMgATP are pH independent. The V/K for Ser-peptide is bell-shaped with pK values of 6.2 and 8.5 estimated. The pH dependence of 1/Ki for Leu-Arg-Arg-Ala-Ala-Leu-Gly is also bell-shaped, giving pK values identical with those obtained for V/KSer-peptide, while the Ki for MgAMP-PCP increases from a constant value of 650 microM above pH 8 to a constant value of 4 mM below pH 5.5. The Ki for uncomplexed Mg2+ obtained from the Mg2+ dependence of V and V/KMgATP is apparently pH independent.     

Effects of low electrolyte media on salt loss and hemolysis of mammalian red blood cells.

Cation loss and hemolysis of various mammalian red cells suspended in isotonic non-electrolyte media were investigated. Sucrose buffered with 10 mM Tris-Hepes, pH 7.4 was used as the non-permeable non-electrolyte. Mammals from which the red cells were derived include the human, guinea pig, rat, rabbit, newborn calf, newborn piglet and pig, all of which contain K as the predominant cation species (HK type) and the dog, cat, sheep and cow, all of which possess Na as the predominant cation species (LK type). Of HK cells, a rapid efflux of K takes place from humans, rats and guinea pigs. Of LK type cells, the dog and cat exhibit an augmented membrane permeability to Na. The governing factors which influence cation permeability are the change in pH, temperature, and ionic strength. In response to increase in pH, the red cells of humans, dogs and cats become more permeable to cations, whereas the red cells of rat and rabbit are unaffected. In response to increase in temperature, HK type cells exhibit augmented K efflux, while the Na loss from the dog and cat cells manifest a well-defined maximum at near 37 degrees C. In all cases, a small substitution of sucrose by an equal number of osmoles of salts results in a dramatic decrease in cation loss. By contrast, the red cells of the rabbit, newborn calf, adult cow, newborn piglet, adult pig and sheep display no discernible increase in ion-permeability under the conditions alluded to above. In some species including the newborn calf, dog, and cat, an extensive hemolysis occurs usually within an hour in isotonic buffered sucrose solution. The osmolarity of sucrose solution affects these cells differently in that as the osmolarity increases from 200--500 mM, hemolytic rates of the calf and dog reach a saturation near 300 mM sucrose, whereas the hemolytic rate of the cat decreases progressively. Common features pertaining to this hemolysis are (1) the intracellular alkalinization process; and (2) the diminution of the cell volume which take place prior to and onset of hemolysis. SITS, a potent anion transport inhibitor, completely protects the cells from hemolysis by inhibiting chloride flux and the concomitant rise in intracellular pH.     

Effect of detergents, trypsin and unsaturated fatty acids on latent loquat fruit polyphenol oxidase: basis for the enzyme's activity regulation

The effects of detergents, trypsin and fatty acids on structural and functional properties of a pure loquat fruit latent polyphenol oxidase have been studied in relation to its regulation. Anionic detergents activated PPO at pH 6.0 below critical micelle concentration (cmc), but inhibited at pH 4.5 well above cmc. This behavior is due to a detergent-induced pH profile alkaline shift, accompanied by changes of intrinsic fluorescence of the protein. Gel filtration experiments demonstrate the formation of PPO-SDS mixed micelles. Partial PPO proteolysis suggest that latent PPO losses an SDS micelle-interacting region but conserves an SDS monomer-interacting site. Unsaturated fatty acids inhibit PPO at pH 4.5, the strongest being linolenic acid while the weakest was gamma-linolenic acid for both, the native and the trypsin-treated PPO. Down-regulation of PPO activity by anionic amphiphiles is discussed based on both, the pH profile shift induced upon anionic amphiphile binding and the PPO interaction with negatively charged membranes.     

pH induced changes in optical activity of guanine nucleosides

Optical rotatory dispersion and circular dichroism have been used to investigate the protonation of guanosine and some of its analogues. An inversion of the principal Cotton effect and the dichroic band is observed below the acid pK. It is suggested that a conformational change from the anti form above the pK to the syn form below the pK occurs. The reasons why this change should occur only in guanosine and not in adenosine are discussed.     

Structure and oxidation-reduction behavior of 1-deaza-FMN flavodoxins: modulation of redox potentials in flavodoxins

Flavodoxins from Clostridium beijerinckii and from Megasphaera elsdenii with 1-carba-1-deaza-FMN substituted for FMN have been used to study flavin-protein interactions in flavodoxins. The oxidized 1-deaza analogue of FMN binds to apoflavodoxins from M. elsdenii and C. beijerinckii (a.k.a. Clostridium MP) with association constants (Ka) of 1.0 x 10(7) M-1 and 3.1 x 10(6) M-1, values about 10(2) less than the corresponding Ka values for FMN. X-ray structure analysis of oxidized 1-deaza-FMN flavodoxin from C. beijerinckii at 2.5-A resolution shows that the analogue binds with the flavin atoms in the same locations as their equivalents in FMN but that the protein moves in the vicinity of Gly 89 to accommodate the 1-CH group, undergoing displacements which increase the distance between position 1 of the flavin ring and the main-chain atoms of Gly 89 and move the peptide hydrogen of Gly 89 by about 0.6 A. The X-ray analysis implies that protonation of normal flavin at N(1), as would occur in formation of the neutral fully reduced species, would result in a similar structural perturbation. The oxidation-reduction potentials of 1-deaza-FMN flavodoxin from M. elsdenii have been determined in the pH range 4.5-9.2. The oxidized/semiquinone equilibrium (E'0 = -160 mV at pH 7.0) displays a pH dependence of -60 mV per pH unit; the semiquinone/reduced equilibrium (E'0 = -400 mV at pH 7.0) displays a pH dependence of -60 mV per pH unit at low pH and is pH independent at high pH, with a redox-linked pK of 7.4. Spectral changes of fully reduced 1-deaza-FMN flavodoxin with pH suggest that this latter pK corresponds to protonation of the flavin ring system (the pK of free reduced 1-deaza-FMN is 5.6 [Spencer, R., Fisher, J., & Walsh, C. (1977) Biochemistry 16, 3586-3593]. The pK of reduced 1-deaza-FMN flavodoxin provides an estimate of the electrostatic interaction between the protein and the bound prosthetic group; the free energy of binding neutral reduced 1-deaza-FMN is more negative than that for binding the anionic reduced 1-deaza-FMN by 2.4 kcal.(ABSTRACT TRUNCATED AT 250 WORDS)     

Erythrocyte hemolysis by detergents--a mathematical model and analysis of the concentration and kinetic curves

A mathematical model of erythrocyte lysis by detergents is developed which takes into consideration the kinetics of detergent binding to plasma membrane. Experimentally obtained sigmoidal kinetic and concentration curves of hemolysis are well described by the model. A comparative study is carried out in terms of the model of hemolytic action for five detergents: Triton X-100, sodium dodecylsulfate, sodium deoxycholate, cetyltrimethylammonium bromide, and cetylpyridinium chloride. The amount of detergent which should be bound to an erythrocyte membrane to induce lysis was found to be roughly the same for all detergents studied. However, detergents vary in their affinity to the membrane. Cetylpyridinium displays the highest affinity (and consequently the highest hemolytic activity), whereas deoxycholate has the least one.     

pH studies on the chemical mechanism of rabbit muscle pyruvate kinase. 1. Alternate substrates oxalacetate, glycolate, hydroxylamine, and fluoride

The decarboxylation of oxalacetate shows equilibrium-ordered kinetics, with Mg2+ adding before oxalacetate. The Ki for Mg2+ increases below a pK of 6.9, corresponding to a ligand of the metal that is probably glutamate, and decreases above a pK of 9.2, corresponding to water coordinated to enzyme-bound Mg2+. Both V and V/KOAA decrease above the pK of 9.2, suggesting that the carbonyl oxygen of oxalacetate must replace water in the inner coordination sphere of Mg2+ prior to decarboxylation. The enzyme-Mg2+-oxalacetate complex must be largely an outer sphere one, however, since the pK of 9.2 is seen in the V profile. The phosphorylation of glycolate or N-hydroxycarbamate (the actual substrate that results from reaction of hydroxylamine with bicarbonate) occurs only above the pK of 9.2, with V/K profiles decreasing below this pH. The alkoxides of these substrates appear to be the active species, replacing water in the coordination sphere of Mg2+ prior to phosphorylation by MgATP. Glycolate, but not N-hydroxycarbamate, can bind when not an alkoxide, since the V profile for the former decreases below a pK of 8.9, while V for the latter is pH independent. Initial velocity patterns for phosphorylation of fluoride in the presence of bicarbonate show saturation by MgATP but not by fluoride. The V/K profile for fluoride decreases above the pK of 9.0, showing that fluoride must replace water in the coordination sphere of Mg2+ prior to phosphorylation. None of the above reactions is sensitive to the protonation state of the acid-base catalyst that assists the enolization of pyruvate in the physiological reaction.     

Mechanism of the activating effect of detergents and chelating agents on the Na, K-ATPase activity of erythrocyte ghosts

The reasons for differences in the Na,K-ATPase activity in rat erythrocyte ghosts obtained by hypoosmotic hemolysis in 10 mM Tris-HCl buffer pH 7.6 in the absence ("Tris-ghosts") and presence ("EDTA-ghosts") were investigated. Structurally different detergents (Triton X-100, Tween-20 and sodium deoxycholate) taken at optimal concentrations increased the enzyme activity in a similar way, i. e., 4-fold in "Tris-ghost" and by 30% in "EDTA-ghosts", the absolute activity of Na,K-ATPase in both preparations being levelled out. In the absence of EDTA, only 50-60% of the maximal enzyme activity could be revealed. Thus, in non-nuclear erythrocyte ghosts the maximal Na,K-ATPase activity can be revealed only upon a combined use of a detergent and chelator. It is concluded that the activating effect of the detergents consisting in the increase of the membrane permeability is realized on the outer surface of the membrane, whereas that of EDTA is localized on its inner surface, which is probably due to the disintegration of the cytoskeleton as a result of attachment of membrane-bound Ca2+.